11 BIOSENSORS RESEARCH IN FIRENZE IN THE LAST 25 YEARS Marco Mascini Dipartimento di Chimica, Università di Firenze This lecture will be dedicated to a survey of the major results and activities realised in the last 25 years in our Group in Firenze. Then starting from the preparation of screen printing electrodes we faced several important analytical problems solved by exploiting the use of bioanalytical elements immobilized over several kind of transducers, screen printed electrodes, but also optical devices and piezoelectrical quartz nanobalances, then realizing and applying the Biosensors technology. Three major applications Area of Biosensors have been persecuted. 1)The Diagnostic area in Medicine, in this case starting from the artificial pancreas problem (glucose sensor ) then the interesting goal to succeed to personalise the dialysis of blood with sensors for urea, uric acid etc. then now recently again by exploiting the aptamers realising sensors for a rapid detection of Activated C Protein, Neopterin, Thyroid-stimulating hormone (TSH) etc. 2) the area of Environment where several PhD students get their degree just exploiting the possibility to detect pesticides of a specific enzyme the Acetylcholinesterase or more recently the evaluation of DNA Damage exploiting DNA from calf thimus and the electrochemical signal of the guanine oxidation on graphite just through the screen printing facility. 3) The area of Food, in this case we considered the quality of food (freshness sensors and/or the presence of genetically modified food), and the detection of food safety like in this case the detection of low concentration of bacteria ( Listeria, Salmonella etc.). Finally the recent advances in the field of molecular recognition with the introduction of Aptamers, now divided in two distinct classes, Oligonucleotides Aptamers and Peptide Aptamers from which we are waiting for new devices more stable, more sensitive more selective! Then the match is over and we can continue to look for! 11

12 BIOSENSORS: MEETING THE DEMAND FOR PERSONAL DIAGNOSTICS Anthony P F Turner IFM, Linköping University, S-58183, Linköping, SWEDEN & Cranfield Ventures Ltd., Cranfield University, Cranfield, Bedfordshire, MK43 0AL, UK The world market for biosensors, in 2009, was just under US$13 billion, with nearly nine tenths of that still accounted for by glucose measurement. This extraordinary dominance by a single biosensor type is driven by the exceptional needs of people with diabetes combined with the success of biosensors in meeting their demands for an appropriate product. The overall in vitro diagnostics market is currently considered to be worth around $40 billion, while recent estimates suggest that the theranostics (companion diagnostics) market is potentially worth a staggering $72 billion. Diabetes is the fastest growing chronic disease in the World, with Asia now home to four of the five largest diabetic populations and 2% of the World s population afflicted. Heart disease and stroke kill around 17 million people each year, accounting for one-third of all deaths globally. By 2020, they will be the leading cause of both death and disability worldwide, with fatalities projected to increase to greater than 24 million by Infectious diseases account for over 16% of worldwide deaths, comprising those associated with poverty such as malaria, HIV/AIDS and TB (1/3 rd of world population infected and 2 million deaths) and epidemic & emerging diseases such as meningitis, cholera, yellow fever, flu and antibiotic resistant infections. Around 12% of deaths worldwide are from cancer (Africa 4%, North America 23%; UK 24%) while approximately 11 million people worldwide are diagnosed each year with cancer (45% are in Asia). Finally, society needs to make adequate provision for the proper care of an aging population, the demographic time bomb, with the proportion of people in the world aged 60 yrs old expected to rise from the current 10%, to 22% by Current global healthcare spending is more than $5 trillion per year and is is growing fast (15% of GDP for USA, 8% of GDP for Europe). These combined needs of the individual with societal and economic pressures will force a paradigm change in the way disease is managed, with the future focus on prevention, diagnosis and less expensive, decentralised technologies; in many cases these will be a matter of individual consumer choice. Home testing has been revolutionised by the introduction of colorimetric test strips, electrochemical biosensors, lateral-flow immunoassays and the recent commercialisation of self-implantable glucose biosensors. The completion of the Human Genome project has enable rapid advances in molecular diagnostics and pharmacogenetics, unraveling new pathways and revealing novel potential biomarkers. While personalised medicine is still in its infancy, the poor efficacy of many current pharmaceuticals is a strong driver for combining diagnostics with therapy. Molecular diagnostics to enable better administration of anticoagulant therapy, predict drug metabolism and detect mutations that identify patients likely to respond to cancer therapy, have already met with notable success. The combination of diagnostics with pharmaceuticals not only furnishes immense clinical benefit, but provides a new financial model to drive forward the development of new sensors. With over 6,000 papers published on biosensors in 2009, a plethora of patents and a myriad of new product development announcements, we see visions of the future including exploitation of nanotechnology, synthetic biology and the creation of plastic bioelectronics. This presentation will seek review this emerging new paradigm in biosensor technology. 12

13 METAL OXIDE NANOWIRES FOR CHEMICAL AND BIO-SENSING G. Faglia, C. Baratto, E. Comini, M. Ferroni, A. Ponzoni, S. Todros, D. Zappa and G. Sberveglieri Sensor Laboratory,CNR- IDASC and Brescia University, Via Valotti,9, Brescia Italy Quasi one-dimensional (1D) semiconductor nanocrystals represent the forefront of today s solid state physics and technology. Top-down methods to fabricate 1D nano-scale structures do not guarantee a sufficiently low dimensionality, the resolution limit of nano-lithographic techniques being around 100 nm. Bottom-up techniques have instead great potentials in terms of crystalline perfection, cost and high productivity. One of the most promising self-assembly method is the socalled evaporation-condensation vapour-solid (VS) and metal catalyst assisted vapour-liquid-solid (VLS) mechanism, proposed by Wagner and Ellis in 1964 for silicon whisker growth [1]. Following these routes quasi 1D nanostructures (so called nanobelts or nanoribbons) of semiconducting oxides of Zn, Sn, In, Cd and Ga have been successfully synthesized first by Pan et al. [2]. Shortly afterwards they have become the focus of intensive investigation as potential building blocks for nanoscale devices and sensors. In these studies the metal oxide NWs are typically not intentionally doped, and the carriers are normally generated by structural defects such as oxygen deficiencies. As a result, the devices behave as 1-D wide band gap semiconductors whose current flow is extremely sensitive to minor surface perturbations. When employed as a chemical gas sensor the performances are influenced by the surrounding environment [3], when employed as biosensor after surface grafting with specific receptors, any biological macromolecules bound to the surface and undergoing a binding event with conformational change or change of a charge state, may perturb the current flow in the nanowires fig. 1(b)- On the other hand, intentional doping can provide semi-metallic behavior, greatly affecting the device properties and yielding new device applications, as electrodes in organic solar cells, field emission sources and thin film transistors. Indeed in deep characterization and functional applications of these innovative electrodes are still in their infancy, a field in which they could represent a breakthrough is electrochemical biosensing in the configuration showed in fig. 1(c). State of the art and recent achievements obtained by using highly reactive metal oxides will be presented. (a) (b) (c) Figure 1. SEM image of 1D nws (a), working principles of semiconducting nanowires based electrical biosensors (b) and of an electrochemical biosensor incorporating semimetallic nanowires electrodes (c) References 1. Wagner, R. S.; Ellis, W. C., Applied Physics Letters, 4, (1964) 2. Pan ZW, Dai ZR, Wang ZL., Science 291: (2001) 3. E. Comini, G. Faglia, G. Sberveglieri, Z Pan, Z. L. Wang, Applied Physics Letters 81, (2002) 13

14 ELECTROCHEMICAL BIOSENSING WITH NANOPARTICLES Arben Merkoçi ICREA & Institut Català de Nanotecnologia, CIN2 (ICN-CSIC), Bellaterra (Barcelona), Spain The need for nucleic acid and protein based diagnostic tests has increased enormously in the last few years and the design of nanoparticles with special optical and electrochemical properties is bringing significant advantages in several fields being diagnostic one of the most important. In this context biosensor technology represents an interesting alternative for the development of efficient, fast, low-cost and user-friendly diagnostic devices. Between different biosensing alternatives the nanotechnology and nanomaterial oriented biosensors represent a very attractive tool for clinical applications. DNA, protein and even cell detections methodologies with interest for diagnostics and based on nonoparticles will be described. The developed devices are based on the use of special platforms which allows their future applications and extension in diagnostic. In addition the nanoparticle based biosensors are being offered as excellent screening alternatives to sophisticate and high cost equipments that require well prepared professionals for their use, including data treatment, prior obtaining of final results with interest for diagnostic and treatment. Figure 1. Schematic (not in scale) of some of the optical and electrical detection alternatives that are being used for DNA, proteins and cells analysis thanks to the use of nanoparticles. SPR: surface plasmon resonance; ICP-MS: Inductively coupled plasma mass spectroscopy. [1]. References A.Merkoçi, Biosensors & Bioelectronics, (in print) 14

15 ORALS 15

16 BIO-CHEMILUMINESCENT MINIATURIZED BIOSENSORS Mara Mirasoli 1, Massimo Guardigli 1, Elisa Michelini 1, Patrizia Simoni 2, Luisa Stella Dolci 1, Luca Cevenini 1, Laura Mezzanotte 1, Angela Buragina 1, Francesca Bonvicini 3, Monica Musiani 3, Aldo Roda 1 1 Department of Pharmaceutical Sciences, University of Bologna, via Belmeloro 6, Bologna, 2 Department of Clinical Medicine, University of Bologna, via Massarenti 9, Bologna 3 Department of Haematology and Oncological Sciences L. e A. Seragnoli, Microbiology Section, University of Bologna, via Massarenti 9, Bologna The Point-Of-Care (POC) approach is based on portable devices suitable to perform the analysis directly where it is required or where the sample is obtained. POC, which can provide significant advantages in the clinical field for its ability to provide accurate diagnosis at the patient bedside, can find applications in other fields, such as critical medicine, bio-terrorism, developing countries, food and environmental analyses, space station and telemedicine. A POC device should combine portability, minimum sample pre-treatment and ability to perform highly sensitive multiplexed assays in a short time. Microfluidic integrated systems relying on biospecific recognition reactions (e.g., immunological reactions, nucleic acids hybridization) and ultrasensitive bio-chemiluminescence (BL-CL) detection techniques represent one of the most promising options 1. Although several miniaturized analytical systems were developed, in most cases the miniaturization of the analytical format was not paralleled by an adequate miniaturization of the overall analytical device (e.g., use of laboratory instrumentation for signal detection). This work describes the development of POC devices exploiting contact BL-CL detection, in which the analytical signal is produced directly in the surface of an imaging light sensor able to localize and quantify the signal. In particular, the analytes are captured and detected in different positions of a transparent solid support, which is in contact, through a tapered fiber optic faceplate, with a portable cooled highly sensitive CCD sensor. Contact imaging, which provides high light collection efficiency, has been exploited in different analytical devices 2. A microfluidics-based device was developed for the diagnosis of parvovirus B19 (B19V) infection. In particular, target B19V DNA is captured by a specific peptide nucleic acid (PNA) probe immobilized on a glass surface, and then revealed by exploiting an enzyme label suitable for CL detection. The limit of detection (50 fmol/ml of B19V DNA) is comparable with that obtained with standard laboratory methodologies, with an overall assay time of 30 min. A new portable biodevice containing genetically engineered BL whole-cell biosensors 3 was developed for multiplexed detection of environmental pollutants (e.g., compounds with hormonelike activity, heavy metals ). The cells were modified with the introduction of the gene encoding for a BL reporter protein (e.g., luciferase), so that its expression is controlled by the ability of the target analyte to interact with a specific regulatory proteins or receptor. The expressed reporter protein can be readily measured and directly related to the analyte concentration in the sample. Different recombinant yeast and bacterial biosensors were immobilized in a modified clear bottom black 384-well microplate to obtain a BL cell array suitable for on-site analysis. The cells array, which can be stored for up to 1 month at 4 C without loosing cell vitality, was characterized by high sensitivity (e.g., a LOD of 0.5 nm for testosterone was obtained with immobilized yeast cell-based biosensor for androgen detection after 1-month storage). The performance of contact imaging is evaluated and compared to that of conventional opticsbased imaging performed with laboratory instrumentation. References 1. M. Magliulo, E. Michelini, P. Simoni, M. Guardigli, A. Roda Analytical and Bioanalytical Chemistry 384 (2006) R.R. Singh, D. Ho, A. Nilchi, G. Gulak, P. Yau, R. Genov, IEEE Transactions on Circuits and Systems I-Regular Papers 57 (2010) E. Michelini, M. Magliulo, P. Leskinen, M. Virta, M. Karp, A.Roda. Clinical Chemistry 51 (2005)

17 ELECTROCHEMICAL AND PIEZOELECTRIC IMMUNOSENSORS FOR DETECTION OF BIOAGENTS Petr Skládal Department of Biochemistry and National Centre for Biomolecular Research, Masaryk University, Kotlarska 2, Brno, Czech Republic Electrochemical sensors and piezoelectric quartz resonators were adopted as robust and sensitive transducers for construction of portable detection systems. The former approach utilized multichannel screen-printed sensors combined with specific antibodies and tracers based on peroxidase as a label [1]. The heterogeneous sandwich assay format used exchangeable sensors combined with an embedded flow system consisting of several miniperistaltic pumps. The piezoelectric immunosensor [2] provided direct measurement of the target bioagents, the sensing part was either used repeatedly after regeneration of the immobilized antibody or discarded in the case of gradually consumed binding capacity. From the construction point of view, both lines of portable detectors ImmunoSMART and QCM-meter - shared several common elements (microcontroller, A/D converters, and pumps), though the sensing parts were different: multipotentiostat and oscillator-counter, custom flow-through cells and biosensors. Detectors were linked to the external computer with software LabTools (common data-management and user interface, individual detector-related plug-in modules). The tested bioagents included the bacterium Francisella tularensis (Ft, belongs to cat. A biowarfare agents, though the LVS variant was employed) and model microbes Escherichia coli and Bacillus atrophaeus. The electrochemical system was able to detect Ft starting at 100 CFU/ml; one assay (incubations with sample and tracer, washing, addition of enzyme substrates, amperometric measurement) was completed within 20 min. The piezoelectric immunosensor with immobilized anti-ft antibody indicated the presence of 10 5 CFU/ml directly within 10 min. Using the immunosensor modified with Ft derived lipopolysaccharides, rapid detection of anti-ft antibodies from mice inoculated with the bioagent (0.1 LD50) was possible shortly (1-3 days) after infection. The E. coli-based bioaerosols were sampled after connection of the ImmunoSMART sensor with a cyclone device SASS These experiments were carried out in a closed chamber and 100 CFU/l in air was successfully detected. The developed biosensor-based devices are promising tools suitable for rapid detection of bioagents as well as other microbial cells and biomolecules outside of laboratory. References 1. Skládal, P., Pohanka, M., Kupská, E., Šafář, B. (2010). Biosensors for Detection of Francisella tularensis and Diagnosis of Tularemia. In: Biosensors, INTECH, Vienna, pp Skládal, P. (2009). Piezoelectric Quartz Crystal Resonators Applied for Immunosensing and Affinity Interaction Studies. In: Biosensors and Biodetection Methods and Protocols, Vol. 2, Humana Press, New York, pp

18 ELECTROCHEMICAL SENSORS, BIOSENSORS AND IMMUNOSENSORS APPLIED IN CLINICAL, FOOD AND ENVIRONMENTAL ANALYSIS. Giuseppe Palleschi, Danila Moscone, Laura Micheli, Giulia Volpe, Francesco Ricci, Fabiana Arduini, Federica Valentini, Silvia Piermarini. Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma Tor Vergata, via della Ricerca Scientifica, Roma The scientific heredity left by Prof. Marco Mascini when he moved from Rome to Florence in 1985 has progressed in the field of electrochemical sensors and has produced several applications in the areas of clinical, food and environmental analysis. This keynote lecture will show some highlights during 25 years of research activity at the University of Rome Tor Vergata and is dedicated to Prof. Marco Mascini. In these years we moved from the first generation of biosensors to second and third one, and from the use of classical sensors to the new, ease to use, cost effective, and versatile screen printed electrodes (SPEs) that resulted suitable for many modifications by the use of mediators and nanostructured materials. However, also classical potentiometric and amperometric sensors, constructed to micrometric scale, have been used for the detection of ph, calcium, potassium, ammonium and oxygen respectively to monitor the growth of cyanobacteria in the Roman Catacombs. Prussian Blue (PB) modified SPEs have been assembled using a new chemical deposition procedure for the selective detection of hydrogen peroxide at low potential. The excellent analytical features of these sensors led to the development of a glucose biosensor that is now the analytical device of the Glucomenday, a new holter for continuous glucose detection in diabetic patients produced by the Menarini Company. Another interesting application of the PB-based sensors is the possible detection of thiols in clinical samples. We have in fact demonstrated that the Prussian Blue not only acts as an electrocatalytic mediator for H 2 O 2 reduction but also for the oxidation of several thiols. We have applied thiese sensors for the detection of thiocholine, a key analyte for the measurement of pesticides, heavy metals such as Hg, and glutathione. PB-SPEs were also applied to monitor hydrogen peroxide during organic reactions in ionic liquids, with the advantage of carrying out environmental friendly reactions, according to the principles of the green chemistry. Electrochemical disposable PB-modified biosensors based on cholinesterase enzyme inhibition have been assembled and used for organophosphorous and carbamic pesticides detection in river water samples and for the detection of chemical warfare agents (Sarin gas). Concerning the use of nanomaterials, Carbon Nanotubes (CN) and Carbon Black (CB) nanoparticles have been used to assemble new sensors and biosensors able to measure, with improved sensitivity, important neurotransmitters such as dopamine, epinephrine and serotonin, biological molecules, as catechol, guanine and tyrosine, inorganic electroactive molecules as ferricyanide, and also hydrogen peroxide and NADH. Functionalized carbon nanofiber-based chemical sensors for the direct electrochemistry of the haemoglobin and myoglobin molecules have been developed. Electrochemical sensors modified with nanostructured polymers have been assembled for the detection of nitrites and ammonia to monitor the chemical potability of water. New nanomaterials such as few layer of functionalized graphene are actually under study for glucose biosensor applications. SPEs have been also modified with films of Bi for the evaluation of lead bioaccumulation in aquatic macrophytic plant Lemna Minor. Also an exhaustive study of milk treatment to measure Pb in this complex matrix using bismuth modified SPE has been successfully carried out. Immunosensors for marine toxins and for mycotoxins have been assembled and applied to detect the aflatoxin M1 in milk and the B1 in cereals. More recently trichothecenes DON, T-2, and HT-2 have been detected in cereals using electrochemical sensors arrays coupled with micromagnetic beads (as support for the immunological chain). Immunosensors for different bacteria detection have been also developed. An interesting application in food analysis has been the assembling of an immunosensor for Salmonella detection 18

19 in different kind of meats with an analysis time of few hours instead of days. Immunosystems for the detection of Botulin toxins and for the rapid identifications of Celiac disease in patients are actually in progress in our research group. A new research line concerns the development of new reagentless, single-step electrochemical DNA sensors (E-DNA). These biosensors have demonstrated to be able to recognize not only the complementary DNA sequence of the immobilized probe, but also other proteins such as anti-dna antibodies, for example, directly in blood serum, permitting to track the progression of autoimmune diseases like systematic Lupus erythematosus, that affects more than five million people worldwide. 19

20 DEVELOPMENT OF NEW ELECTRONIC BIOSENSORS BASED ON ORGANIC THIN FILM TRANSISTORS Maria Magliulo 1, Maria Daniela Angione 1, Serafina Cotrone 1, Antonia Mallardi 2, Roberto Gristina 3, Gerardo Palazzo 1, Francesco Palmisano 1, Luisa Torsi 1 1 Dipartimento di Chimica, Università degli studi di Bari, via Orabona 4 2 Istituto per i Processi Chimico-Fisici (IPCF), CNR, Università degli studi di Bari, Via Orabona 4 3 Istituto di Metodologie Inorganiche e dei Plasmi (IMIP), CNR Most of biological sensing techniques are based on optical detection principles that are highly sensitive and specific but very difficult to miniaturize. These techniques also require multiple reagents, long preparative steps and complex data analysis. Organic thin film transistors (OTFTs) can offer an alternative to overcome some of the optical biosensors drawbacks. Simple and lowcost fabrication techniques, miniaturization, multi-parametric responses, signal amplification and label-free detection are the main features of OTFT biosensors 1,2. Particularly, OTFT technology can be implemented to develop label-free DNA or bio-affinity sensor chips, having a field-effect transport directly coupled to a bio-sensing process, useful to high-throughput testing and point-ofcare applications 3. The development of new structures that can provide the full integration in OTFT devices of biological recognition elements such as antibodies or other proteins to confer specificity is the main goal of this study. The coupling of the OTFT device and the biological recognition system is actuated by assembling supramolecular structures that integrate biomolecules deposited directly in the OTFT active layer. Bio-probes are immobilized on the sensors surface using either conventional procedures and more innovative strategies based on the use of phospholipid bilayers. The efficiency of the immobilization procedures is evaluated by fluorescence imaging techniques. Preliminary results obtained by using the anti-biotin/biotin/streptavidine reagent systems will be presented. The possibility to develop OTFT biosensors capable of offering enhanced sensing performance, particularly in terms of sensitivity and bias control, will be also discussed. The reported technology hold the potential to display a great versatility allowing for a wide range of applicability to many different assays by just binding the right bio-recognition element for the analyte of interest. Besides, the label-free assay format ensures a reduction of assay time and reagents consumption with respect to methods relying on labeled molecules References 1. N.A. Sokolov, M.R. Roberts and Z. Bao, Materials today 12 (2009) L. Torsi, G.M. Farinola, F. Marinelli, M.C. Tanese, O. Hassan Omar, L. Valli, F. Babudri, F. Palmisano, P.G. Zambonin, F. Naso, Nat Mater 7 (2008) F. Yan, S. M. Mok, J. Yu, H. L.W. Chan and M. Yang, Biosens. Bioelectron. 24 (2009)

21 Au NANOPARTICLES IN ELECTROANALYSIS Chiara Zanardi, Fabio Terzi, Barbara Zanfrognini, Laura Pigani, Renato Seeber Dipartimento di Chimica, Università di Modena e Reggio Emilia, via G. Campi 183, Modena, Metal nanoparticles (NPs) have recently assumed a prominent role in the development of amperometric sensors [1-4]. From a general point of view, NPs can be defined as the individual constituents of extremely fine powder (1-100 nm size), being usually considered transitional between the clusters, i.e. aggregates of few atoms, and the bulk scale. NPs possess a polyhedric shape, which makes a large fraction of atoms be located in the correspondence to such defects as vertexes and edges. This especially holds in the case of real NPs, typically under 20 nm size. Hence, metals under the form of NPs possess (electro)catalytic properties by far superior to those of bulk systems, which may increase the resolution of the response pattern, as well as the number of detectable/quantifiable analytes. Moreover, NPs possess a particularly high surface/volume ratio: when considering NP systems as electrode modifiers, an increased surface area is obtained with respect to bulk materials, often resulting in improved sensing sensitivity. Such a nanostructure may also increase the anchoring stability of elements sensitive toward species of biological interest and to facilitate the charge transfer. This communication aims at critically summarising the most recent advances in the development of amperometric sensing systems based on AuNPs, ranging from chemical sensors for different inorganic and organic species to biosensors. Attention will be also devoted to the comparison of the synthetic approaches followed for obtaining nanostructured modified surfaces with the properties sought. The different techniques employed in the characterisation of nanostructured surfaces obtained after the deposition of AuNPs will be also outlined. Riferimenti 1. D. Hernandez-Santos, M. B. Gonzalez-Garcia, A. Costa GarcÌa, Electroanalysis 14, (2002) S. Guo, E. Wang, Anal. Chim. Acta 598 (2007) C. M. Welch. R. G. Compton, Anal. Bioanal. Chem. 384, (2006) F. W. Campbell, R. G. Compton, Anal. Bioanal. Chem. 396, (2010)

24 Abs (a.u.) 1645 Abs (a.u) Abs 1342 Abs DNA-BASED BIOSENSOR, AND SURFACTANT TOXICITY: A FT-IR INVESTIGATION Francesca Cugia 1, Andrea Salis 1, Maura Monduzzi 1,and Marco Mascini 2 1 Dipartimento di Scienze Chimiche, Università degli studi di Cagliari, S.S. 554 bivio Sestu, Monserrato 2 Dipartimento di Chimica, Università di Firenze, via della Lastruccia 3, Sesto Fiorentino (FI) In the present work a DNA biosensor was used for the determination of the toxicity of different kinds of common surfactants. The intensity of the guanine oxidation peak, measured through Square Wave Voltammetry usually quantifies (G% with respect to a buffer solution) the interactions between DNA and the eventually genotoxic substances. All surfactants caused a decrease of the guanine peak, in particular the nonionic surfactants were highly toxic (G% < 40%,) followed by the moderately toxic anionic surfactants (G% ~ 50) and cationic surfactants (G% ~ 55%). Some selected surfactants were investigated both in sea water and tap water, and data were compared to those obtained in acetate 0,20 0,20 buffer. In several cases it was observed that toxicity decreases with increasing concentration DNA biosensor as a consequence of surfactant self-assembly. Indeed, the self-assembly 0,15 process competes with 0,15 the interaction with DNA. Hence the toxicity can qualitatively be related with the self-assembly 0,10 0,10 behavior of the different surfactants in the different media. The interaction between surfactants and Calf Thymus DNA in solution 0,05 and adsorbed on the sensor 0,05 surface was investigated through FTIR-BioATR and FTIR-ATR spectroscopy respectively. 0,00 0, DNA in solution a DNA biosensor b DNA biosensor-ctac c Abs (a.u.) wave number (cm -1 ) wave number (cm -1 ) wave number (cm -1 ) Figure 1. a) DNA spectra in solution; b) DNA biosensor spectra; c) DNA biosensor spectra after interaction with CTAC surfactant; Significant specific modification of DNA bands appear as a result either of the immobilization process on the graphite solid surface or after the interaction with the surfactant as shown in figure1. 24

25 NON-INVASIVE PRENATAL DIAGNOSIS OF -THALASSEMIA BASED ON FETAL DNA FROM PERIPHERAL BLOOD AND SEQUENCING OF -GLOBIN PCR PRODUCTS Giulia Breveglieri, 1,2 Alessia Finotti, 2 Giordana Feriotto, 2 Francesca Salvatori, 1 Roberta D Agata, 3 Giuseppe Spoto, 3 Roberto Gambari 1,2 1 Department of Biochemistry and Molecular Biology, University of Ferrara, Ferrara, 2 Biotechnology Center, University of Ferrara, Ferrara 3 Dipartimento di Scienze Chimiche, Università di Catania, Catania We have determined the efficacy of sequencing PCR products obtained using as target free genomic DNA from peripheral blood enriched for low molecular weight DNA, which contains high quantity of free fetal DNA (ffdna). Genomic DNA was isolated from peripheral blood of a pregnant woman exhibiting a 39/ + IVSI-110 genotype. After DNA isolation using QIAamp DNA Blood Mini Kit (Qiagen), genomic DNA was separated by agarose gel electrophoresis, the low molecular weight DNA purified and PCR performed using primers amplifying a genomic - globin DNA region containing the IVSI-1, + IVSI-6, + IVSI-110 and 39 thalassemia mutations. The electropherograms obtained after sequencing of PCR products demonstrate that the samples enriched of ffdna display a high + IVSI-110/wt peak ratio, while the 39/wt ratio was low. On the contrary, the + IVSI-110/wt and 39/wt peak ratios were found to be balanced in the PCR product from the maternal genomic DNA. This finding suggests a + IVSI- 110/wt genotype of the fetus. In order to determine the detection limit of the technology, DNA samples mimicking fetal DNA were diluted with maternal DNA and the same analysis performed. The results obtained suggest that the genotype of the ffdna should be detectable when at least 15% of fetal DNA is present in the preparation. Future perspectives of this approach should consider the combination between ffdna isolation and other analytical techniques, including the use of Surface Plasmon Resonance (SPR)-based strategies. Our group has in fact demonstrated that SPR and Biospecific Interaction Analysis are efficient tools for detecting point mutation of -thalassemia. 1,2 Interestingly, recent developments of surface plasmon resonance technology, at least in theory, are compatible with the possibility to perform diagnostic analyses on PCR-free unamplified genomic DNA (D Agata et al., manuscript in preparation), opening novel frontiers in molecular diagnosis, including non-invasive prenatal tests. References 1. G. Feriotto, G. Breveglieri, S. Gardenghi, G. Carandina, R. Gambari. Molecular Diagnosis 8 (2004) G. Feriotto, G. Breveglieri, A. Finotti, S. Gardenghi, R. Gambari. Laboratory Investigation 84 (2004)

26 SPRI FOR THE DETECTION OF GENOMIC DISORDERS IN UNAMPLIFIED HUMAN GENOMIC DNA Roberta D Agata, 1 Giulia Breveglieri, 2 Laura Maria Zanoli, 3 Monica Borgatti, 2 Alessia Finotti, 2 Giuseppe Spoto, 1,4 Roberto Gambari 2 1 Dipartimento di Scienze Chimiche, Università di Catania, Catania, 2 Biotechnology Center, University of Ferrara, Ferrara, 3 Scuola Superiore di Catania, Università di Catania, Catania, 4 Istituto Biostrutture e Bioimmagini, CNR, Catania DNA sensing is expected to be significantly improved by using simple and economic detection protocols which require minimal DNA modifications and provide enhanced signal amplification. In this perspective, the direct analysis of non amplified genomic DNA appears an excellent costeffective alternative to the current available methods that can be achieved by using ultrasensitive DNA detection technologies. Recently, we have shown that an ultrasensitive detection of non-amplified genomic DNA containing a target sequence as a minor component can be obtained by using a nanoparticle-enhanced Surface Plasmon Resonance Imaging (SPRI)-based detection protocol and surface-immobilized peptide nucleic acids (PNA) probes. 1,2 In this work we demonstrate that point mutations in human genome can be also detected by adopting the nanoparticle-enhanced SPRI protocol based on the hybridization of sonicated nonamplified genomic DNA to PNA probes. The approach has been successfully implemented for the identification of the β 39 (C>T) thalassemia point mutation of the human beta-globin gene, demonstrating that the method provides a highly specific and cost-efficient approach for the detection of point mutations. To the best of our knowledge, the method is the first reporting the possible use of SPRI in detecting human genetic mutations without PCR-mediated amplification. The observed detection efficiency allows us to claim that unamplified genomic DNA from low numbers of cells (few thousands) might be analyzed by using a very simple protocol, thus avoiding enzymatic treatment or complex thermal cycling of the genomic material. The efficiency is compatible with a PCR-free detection of human mutations starting with fetal-dna enriched samples from peripheral blood in procedures aimed at the non-invasive prenatal diagnosis. References 1. R. D Agata, R. Corradini, G. Grasso, R. Marchelli, G. Spoto, ChemBioChem 9 (2008) R. D Agata, R. Corradini, C. Ferretti, L. Zanoli, M. Gatti, R. Marchelli, G. Spoto, Biosensors and Bioelectronics 25 (2010)

27 ELECTROCHEMICAL GENOSENSOR ARRAY FOR COELEIAC DISEASE PREDISPOSITION ANALYSIS Valerio Beni 1, Hamdi Joda 1, Deirdre Cournane 2, Ioanis Katakis 1 and Ciara K. O Sullivan 1, 3 1 Departament d Enginyeria Quimica, Universitat Rovira i Virgili, Avinguda Països Catalans, 26, Tarragona, Spain, 2 Department of Biochemistry, Universitry College Cork, Cork, Ireland 3 Institucio Catalana de Recerca i Estudis Avançats, Passeig Lluis Companys 23, 08010, Barcelona, Spain Coeliac disease (CD) is a small intestinal inflammation triggered by the intake of gluten, a protein present in certain cereals [1]; this has a prevalence of about 1% in the European population [2]. CD has been shown to affect only genetically predisposed individuals; strong relation between this disease and Human Leukocyte Antigens (HLA) has been proved, with 95% of the CD patients carrying HLA DQ2 genotype and ca. 3% carrying DQ8 genotype [3]. DQ2 and DQ8 negative individuals have been shown to be very unlikely to develop CD. Electrochemical genosensors are low cost, easy to use, rapid, suitable for miniaturization and easy to integrate devices. The aim of the reported work is the development of an electrochemical genosensors array for the rapid and cost effective typing of Coeliac disease associated HLA genes. The proposed sensing platform is based on the Sequence Specific Oligonucleotides Probes (SSO) approach where discrimination is made possible by the use of specific recognition probes. Electrochemical transduction of the probe/target hybridisation event is performed by the use of enzymatic label; this allowed a limit of detection of ca. 1 nm. ELONA, SPR and electrochemical evaluations of different probe designs, for the detection of CD associated alleles (DQA1*0201, DQA1*03, DQA1*050101/DQA1*0505, DQB1*02 and DQB1*03*), are reported. Optimal designs of probes for DQA1*0201 and DQA1*03 and DQA1*050101/DQA1*0505 has been achieved while further optimizations are required for DQB1*02 and DQB1*03* specific probes. Finally preliminary results on the analysis of PCR product will be also presented. Reference 1. M. N. Marsh QJM. 88, (1995), C. Dube, A. Rostom, R. Sy, A. Cranney, N. Saloojee, C. Garritty, M. Sampson, L. Zhang, F Yazdi, V. Mamaladze, I. Pan, J. MacNail, D. Mack, D. Patel, D. Moher, Gastroenterology 128 (2005), S L. M. Sollid, Annu Rev Immunol 18 (2000),

28 A MIP-MODIFIED CARBON STRIP CELL AS SELECTIVE ELECTROCHEMICAL SENSOR FOR FIELD DETERMINATION OF TNT Maria Pesavento, Girolamo D Agostino, Giancarla Alberti, Raffaela Biesuz Dipartimento di Chimica Generale, Università di Pavia,Via Taramelli Pavia-Italy, Aromatic nitroderivatives can be determined electrochemically since nitro groups are reduced at relatively high potentials. Several working electrodes have been proposed for the determination 1 included planar screen-printed carbon electrodes 2, 3. These are very convenient devices for field determinations, particularly if integrated in strip cells consisting of planar electrodes, working, auxiliary and reference electrodes, all integrated in the same solid support. The resulting cell is miniaturized in comparison to the usual cells and is suitable for on site applications. Moreover very small solution volumes, even a drop of liquid, are required. Strip cells can be prepared in a reproducible way and at low cost by the screen printing technology. For these reasons strip cells are suitable for in situ determinations of electroactive substances, for example heavy metals 4. They have been used as working electrodes for detection of aromatic nitroderivatives 2, 3 and other organic electrochemically active substances. However, as usual in electrochemical methods, the poor selectivity can be a problem. In the sensor here proposed a good selectivity was achieved by cell modification with a thick layer of ion exchanging MIP. A MIP (Molecular Imprinted Polymer) specific for trinitrotoluene (TNT) was obtained by the molecular imprinting technology methods. Methacrylic acid (MAA) was the functional monomer and ethylene glycol dimethacrylate (EGDMA) was at the same time the cross-linker and the solvent 5. The liquid monomeric mixture was placed all over the zone of the strip containing the electrodes, and the polymerization was carried out at high temperature (80 C) The solid polymeric film formed in this way covers all three electrodes. The electrolytic solution in contact with the cell is the MIP itself, in which the mobile cations are the counter ions of the negatively charged carboxylic groups fixed in the mainframe of the polymer. It has been found that the charge current is high in MIP-modified strip cell even in pure water, or in the air. Thus it is expected that measurements can be done directly in low ionic strength solution, as for example in ground waters. The possibility of performing measurements without any treatment or modification of the sample was investigated, considering in particular the effect of acidity and ionic composition. Some results are reported in Figure 1, in which peak currents obtained at different conditions are reported. It is seen that a signal is obtained at all the considered conditions, so that the presence of TNT can always be determined, even though different detection limits are attainable. The sensitivity is noticeably affected by the conditions. Figure 1- ip (DPV) of 1 ppm TNT in A on bare and MIP modified carbon electrode at different conditions On the basis of the obtained results the conditions under which highest sensitivity was obtained were assessed. References 1. J. Wang, Electroanalysis 19 (2007) J. Wang, F. Lu, D. MacDonald, J. Lu, M.E.S. Ozsoz, K.R. Rogers, Talanta 46 (1998) K.C. Honeychurch, J.P. Hart, P.R.J. Pritchard, S.J. Hawkins, N.M. Ratcliffe, Biosensors and Bioelectronics 19 (2003) I. Palchetti, S. Laschi, M. Mascin, Anal. Chim. Acta 530 (2005) G. D Agostino, G. Alberti, R. Biesuz, M. Pesavento, Biosensors and Bioelectronics 22 (2006)

29 SOLID SUPPORTED MEMBRANES AS A BIOSENSOR SYSTEM FOR THE INVESTIGATION OF MEMBRANE TRANSPORTERS Francesco Tadini-Buoninsegni, Gianluca Bartolommei, Maria Rosa Moncelli Department of Chemistry Ugo Schiff, University of Florence, Via della Lastruccia 3, Sesto Fiorentino, Italy, A biosensor technology, based on a solid supported membrane (SSM), is currently employed to detect charge movements in electrically active membrane transporters (1). The SSM represents a model system for a lipid bilayer membrane with the additional benefit of being so mechanically stable that solutions may be rapidly exchange at the surface (2). Proteoliposomes or native membranes (vesicles or fragments) incorporating the transport protein can be adsorbed on a SSM and activated using a rapid substrate concentration jump. The substrate jump induces charge displacement within the transport protein, resulting in a current transient which can be detected in the external circuit (1,3). Therefore, the SSM serves two purposes at once, i.e. offering an adhesive surface to the adsorbed membrane entities and functioning as a transducer of a biosensor system. An advantageous feature of the SSM method consists in providing an aqueous environment on both sides of the membrane for the incorporated transport proteins. In addition, adsorption of proteoliposomes or membranes allows a number of primary and secondary active membrane transporters to be immobilized on the SSM in a simple spontaneous process. Very recently, an automated version of the traditional SSM method has been developed which is adapted to the requirements of industrial drug screening (4,5). The financial support of Ente Cassa di Risparmio di Firenze and Ministero dell Istruzione, dell Università e della Ricerca (PRIN Project) is gratefully acknowledged. References 1. P. Schulz, J.J. Garcia-Celma, K. Fendler Methods 46 (2008) J. Pintschovius, K. Fendler Biophys. J. 76 (1999) F. Tadini-Buoninsegni, G. Bartolommei, M.R. Moncelli, K. Fendler Arch. Biochem. Biophys. 476 (2008) B. Kelety, K. Diekert, J. Tobien, N. Watzke, W. Dörner, P. Obrdlik, K. Fendler Assay Drug Dev. Technol. 4 (2006) S. Geibel, N. Flores-Herr, T. Licher, H. Vollert J. Biomol. Screening 11 (2006)

30 INHIBITION OF MEMBRANE TRANSPORTERS: QUANTITATIVE INFORMATION OBTAINED THROUGH A BIOSENSOR TECHNOLOGY Gianluca Bartolommei, Francesco Tadini-Buoninsegni, Maria Rosa Moncelli Department of Chemistry Ugo Schiff, University of Florence, Via della Lastruccia 3, Sesto Fiorentino, Italy, Membrane transporters are devoted to ion translocation through a lipid membrane phase. Among them, fundamental physiological roles are held by the ion pumps Ca-ATPase and Na,K-ATPase, prominent members of the P-type ATPases family (1). BioElectroLab has a wide expertise in the study of ion transport by these proteins through a biosensor technology based on solid supported membranes (2). Our attention has been recently focused on the inhibition of ion pumps by molecules of potential pharmacological interest (3) and xenobiotics (4). Molecules like thapsigargin and cyclopiazonic acid belong to high (nanom) affinity inhibitors of the Ca-ATPase, whereas curcumin and clotrimazole are medium (microm) affinity inhibitors of both Ca-ATPase and Na,K-ATPase. Moreover, the toxic heavy metal Pb 2+, that poses a major public health problem, is able to inhibit Na,K-ATPase activity in the low micromolar range. Frequently, inhibitors confine an ion pump in an inactive conformation. Our experimental technique can provide useful information about the inhibition mechanism at molecular level, as well as quantitative data about the affinity of the molecule (or ion) for the enzyme through a half-inhibition constant value (K i ). Here we present a summary of results obtained in our laboratory for several compounds together with some preliminary data from the current research. Financial support of Ente Cassa di Risparmio di Firenze and of MIUR (PRIN Project) is gratefully acknowledged. References 1. Moller, J. V., Juul, B.le Maire, M. Biochim Biophys Acta 1286 (1996), Tadini-Buoninsegni, F., Bartolommei, G., Moncelli, M. R.Fendler, K. Arch Biochem Biophys 476 (2008), (review) 3. Tadini-Buoninsegni, F., Bartolommei, G., Moncelli, M. R., Tal, D. M., Lewis, D.Inesi, G. Mol Pharmacol 73 (2008), Bartolommei, G., Gramigni, E., Tadini-Buoninsegni, F., Santini, G.Moncelli, M. R. Biophys J (2010), in press. 30

31 GOLD NANORODS FOR LSPR BASED BIOSENSORS Ilaria Mannelli 1,2, Núria Tort 1,2, Fátima Fernández 1,2, J.-Pablo Salvador 2,1, Borja Sepúlveda 3, Laura M. Lechuga 3, M.-Pilar Marco 1,2 1 Applied Molecular Receptors Group (AMRg), Institute for Advanced Chemistry of Catalonia (IQAC) of the Spanish Council for Scietific Research (CSIC) 2 Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) 3 Nanobiosensors and Molecular Nanobiophysics Group, Research Centre in Nanoscience and Nanotechnology (CIN2) CSIC-ICN Noble metal nanoparticles have attracted great attention due to their unique optical properties, in particular the localized surface plasmon resonance (LSPR). Because of the LSPR phenomenon, the nanoparticle extinction spectrum is affected by the particle size and shape as well as the constituting material and the surrounding dielectric media. Several analytical and bioanalysis applications, in which the nanorod optical properties are exploited, have been presented in the last years. Within them, various LSPR based biosensor formats have been proposed. The identification of an analyte could be accomplished by recording the shift of their LSPR peak. A specific bioreceptor is immobilised on the nanoparticle surface and the analyte/receptor biomolecular interaction modifies the dielectric properties of the surrounding medium, with consequent changes in the resonance peak. In view of this, a study for the development of an immunosensor for anabolic androgenic steroids (AAS) based on the use of gold nanoparticles has been performed. In recent years the assumption of dietetic supplements and drugs is drastically increased because of current social and cultural habits. Most of these substances are included in the list of prohibited compounds of the World Anti- Doping Agency and within them the AAS. A World Anti-Doping Code has been drawn up in order to coordinate effective anti-doping programs. In this context, the demand of new rapid, efficient and throughput detection systems is always present. In this work, first, a surface chemistry for conjugating antibodies specific for AAS to gold nanorods has been optimized and the obtained bioconjugates have been characterized. Moreover, the analytical performances of the bioconjugates are under evaluation in a capture competitive sensor format, in which the competitors are directly immobilised onto the sensor surface and the antigenantibody recognition took place when the specific antibodies labelled with gold nanorods were introduced. Preliminary results demonstrated that the detection signal is due to the appearance of a plasmon peak only where specific antigen/antibody interaction occurs and that it is related to the different amounts of nanorods interacting with the surface. In future, the identification of several prohibited substances can be accomplished by using bioconjugate nanoparticles differing in shape and size (label-encoded microarray) or by the location in the microarray (site-encoded microarray). Figure 1. Gold nanorods addition over functionalized surface produces a change in the intensity of the plasmon peak. 31

32 BIOLUMINESCENT GENETICALLY ENGINEERED CELLS FOR BIOSENSING APPLICATIONS Luca Cevenini a, Elisa Michelini a,b, Laura Mezzanotte a, Andrea Coppa a, Aldo Roda a,b a Department of Pharmaceutical Sciences, University of Bologna, Bologna, Italy; b INBB, Istituto Nazionale di Biostrutture e Biosistemi, Roma, Italy Bioluminescence (BL) has revealed an extremely useful analytical tool enabling ultrasensitive detection in biotechnological applications. Genetically engineered cells (bacteria, yeasts, or mammalian cells) able to produce a BL signal in response to a target analyte represent powerful bioanalytical tools for environmental, medical, and food analysis 1. Their principal advantages are high sensitivity, rapidity, low cost, wide dynamic range, and adaptability to high-throughput screening. Nevertheless some important issues have still to be addressed to make these biosensing systems true analytical biosensors, such as portability and reliability to enable their use for on field monitoring and point-of-care analysis. To address this issue, a new polymeric matrix was developed to encapsulate and keep cells alive for long periods of time in order to obtain ready-to-use portable devices. Different recombinant yeast, bacterial and mammalian biosensors were immobilized to obtain a bioluminescent cell array that can be stored for up to 1 month at 4 C without loosing cell vitality 2. These biosensing systems were obtained by introducing into bacterial or yeast cells a vector encoding for a receptor or a regulatory protein (e.g., human androgen/estrogen receptors, regulatory proteins involved in heavy metal-induced transcription ) and a vector with a BL protein (a luciferase) whose expression is controlled by activation of receptors or regulatory proteins. The expression of the reporter protein can be readily measured and directly related to the analyte bioavailability in the sample. The cell array is placed in contact, through an optical taper, with an imaging light sensor, a portable CCD camera able to localize and quantify the luminescent signal. Limit of detections comparable to those reported with liquid cultures were obtained with S.cerevisiae cells responding to androgens 3 and with different bacterial strains responding to heavy metals (cupper, zinc, lead) after 2 weeks storage of immobilized cells at 4 C (e.g., for the strain E. coli MC1061-cueR/pDNPcopAluc the LOD for cupper was 2 x 10-6 M with a dynamic range of 2 x x 10-3 M). In addition, multiplexing capacity (high content screening) should be provided in order to reduce the number of analyses to obtain a complete sample profile. The recent availability of new reporter genes with improved BL properties, together with technical improvements, prompted the development of multiplexed cell-based assays using more BL proteins (e.g., green- and redemitting luciferases, secreted luciferases or calcium sensitive photoprotein aequorin) under the regulation of constitutive and inducible promoters in the same cell. In particular multi-color bioassays were developed for the simultaneous monitoring of different targets (e.g., cyp3a4 and cyp7a1) together with cell vitality. This allowed to measure separate targets within the same cell with good precision (intra-assay and inter-assay CV below 13%). References 1. E. Michelini, M. Magliulo, P. Leskinen, M. Virta, M. Karp, A.Roda. Clin Chem. (2005) 51:1995-8E. 2. E. Michelini, A. Roda, L.S. Dolci, L. Mezzanotte, L. Cevenini. Patent RM2009/A E. Michelini, L. Cevenini, P. Mezzanotte, M. Leskinen, M. Virta, A. Karp, Roda, Nat Protoc 3 (2008)

33 DEVELOPMENT AND VALIDATION OF AMPEROMETRIC IMMUNOSENSORS BASED ON NANOBIOCOMPOSITE MATERIALS FOR THE DETERMINATION OF ALPHA-FETOPROTEIN IN SERUM Marco Giannetto, Maria Careri, Alessandro Mangia, Giovanni Mori, Laura Mori Dipartimento di Chimica Generale ed Inorganica, Chimica Analitica Chimica Fisica Università di Parma, Viale G.P. Usberti, 17/A, Parma Immunosensors have become of special interest in several fields, one of their most important applications being the measurement of compounds of clinical or forensic interest in human serum. The present work deals with the development and validation of a simple to use and rugged immunosensor based on the working principle of the ELISA for the determination of alphafetoprotein (AFP) in serum. The peculiar properties of nanobiocomposite materials based on gold nanoparticles were exploited for the immobilization of the Antibody/Antigen/Antibody-HRP sandwich on a glassy carbon (GC) electrode surface to fabricate an amperometric biosensor. The electrochemical deposition of nanogold on GC electrodes, associated with the use of monomeric thionin (redox mediator), dissolved in the reading solution allowed to provide a simple and reliable approach. Amperometric reading was performed by cyclic voltammetry (CV) measurements carried out with sandwich functionalized electrodes in a buffered solution (ph=6.8) of thionin acetate. Two-way analysis of variance (ANOVA) was applied to find the optimal concentration of the solutions used for the incubation of the antibodies. The sensor was validated in serum assessing stability of the immunocomplex, linearity of response, limits of detection (3.7 ng/ml) and quantitation (11 ng/ml), precision (intra- and intersensor repeatability) and recovery rate (103%).The stability of the GC/Ab functionalized substrate was demonstrated over one month, showing variation coefficients below 5%. Experiments with real samples of clinical interest were also carried out 1. = Studies aimed to the amplification of the response by means of dendrimer-based linking of the catching antibody were also carried out showing interesting findings. For this purpose a self-assembled monolayer of 2-aminoethanethiol (AET) was deposed onto nanogold, and PAMAM G1.5 dendrimers were then bound to the linked AET. Preliminary results obtained with dendrimerlinked antibody show an improvement of the performance in terms of reduced blank signal and widened dynamic range of response Riferimenti 1. M. Giannetto, L. Elviri, M. Careri, A. Mangia, G. Mori, Biosens. Bioelectron, (submitted) 33

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